Tick Salivary Cholinesterase: A Probable Immunomodulator of Host-parasite Interactions.

The southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), is the most economically important cattle ectoparasite in the world. Rhipicephalus microplus and Rhipicephalus annulatus (Say) continue to threaten U.S. cattle producers despite eradication and an importation barrier based on inspection, dipping of imported cattle in organophosphate (OP) acaricide, and quarantine of infested premises. OP acaricides inhibit acetylcholinesterase (AChE), essential to tick central nervous system function. Unlike vertebrates, ticks possess at least three genes encoding AChEs, differing in amino acid sequence and biochemical properties. Genomic analyses of R. microplus and the related tick, Ixodes scapularis, suggest that ticks contain many genes encoding different AChEs. This work is the first report of a salivary cholinesterase (ChE) activity in R. microplus, and discusses complexity of the cholinergic system in ticks and significance of tick salivary ChE at the tick-host interface. It further provides three hypotheses that the salivary ChE plausibly functions 1) to reduce presence of potentially toxic acetylcholine present in the large bloodmeal imbibed during rapid engorgement, 2) to modulate the immune response (innate and/or acquired) of the host to tick antigens, and 3) to influence transmission and establishment of pathogens within the host animal. Ticks are vectors for a greater number and variety of pathogens than any other parasite, and are second only to mosquitoes (owing to malaria) as vectors of serious human disease. Saliva-assisted transmission (SAT) of pathogens is well-known; however, the salivary components participating in the SAT process remain to be elucidated.

Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): construction, expression and biochemical properties of the G119S orthologous mutant.

BACKGROUND: Phlebotomus papatasi vectors zoonotic cutaneous leishmaniasis. Previous expression of recombinant P. papatasi acetylcholinesterase (PpAChE1) revealed 85% amino acid sequence identity to mosquito AChE and identified synthetic carbamates that effectively inhibited PpAChE1 with improved specificity for arthropod AChEs compared to mammalian AChEs. We hypothesized that the G119S mutation causing high level resistance to organophosphate insecticides in mosquitoes may occur in PpAChE1 and may reduce sensitivity to inhibition. We report construction, expression, and biochemical properties of rPpAChE1 containing the G119S orthologous mutation. METHODS: Targeted mutagenesis introduced the G119S orthologous substitution in PpAChE1 cDNA. Recombinant PpAChE1 enzymes containing or lacking the G119S mutation were expressed in the baculoviral system. Biochemical assays were conducted to determine altered catalytic properties and inhibitor sensitivity resulting from the G119S substitution. A molecular homology model was constructed to examine the modeled structural interference with docking of inhibitors of different classes. Genetic tests were conducted to determine if the G119S orthologous codon existed in polymorphic form in a laboratory colony of P. papatasi. RESULTS: Recombinant PpAChE1 containing the G119S substitution exhibited altered biochemical properties, and reduced inhibition by compounds that bind to the acylation site on the enzyme (with the exception of eserine). Less resistance was directed against bivalent or peripheral site inhibitors, in good agreement with modeled inhibitor docking. Eserine appeared to be a special case capable of inhibition in the absence of covalent binding at the acylation site. Genetic tests did not detect the G119S mutation in a laboratory colony of P. papatasi but did reveal that the G119S codon existed in polymorphic form (GGA + GGC). CONCLUSIONS: The finding of G119S codon polymorphism in a laboratory colony of P. papatasi suggests that a single nucleotide transversion (GGC → AGC) may readily occur, causing rapid development of resistance to organophosphate and phenyl-substituted carbamate insecticides under strong selection. Careful management of pesticide use in IPM programs is important to prevent or mitigate development and fixation of the G119S mutation in susceptible pest populations. Availability of recombinant AChEs enables identification of novel inhibitory ligands with improved efficacy and specificity for AChEs of arthropod pests.

Twenty-hour growth hormone secretory profiles after aerobic and resistance exercise.

INTRODUCTION: The pulsatile secretion pattern of growth hormone (GH) is an important parameter of GH action at peripheral tissues, and more information is needed on how exercise impacts GH secretion. This study hypothesized that both aerobic and resistance exercise would exhibit dose-response relationships with respect to exercise duration and 20-h postexercise GH secretion. METHODS: Eight healthy men randomly completed five separate conditions: 1) control (no exercise; CON), 2) a moderate-duration (1-h) aerobic exercise session (MA), 3) a long-duration (2-h) aerobic exercise session (LA), 4) a moderate-duration (1-h) resistance exercise session (MR), and 5) a long-duration (2-h) resistance exercise session (LR). Exercise intensity, diet, sleep, and physical activity were strictly controlled during each condition, and blood was sampled postexercise every 20 min for 20 h, and GH secretion parameters were analyzed via cluster and deconvolution analyses. RESULTS: Only the 2-h aerobic exercise bout resulted in a significant amplification of GH secretion as evidenced by increases in GH burst peak amplitude (∼100%), basal GH secretion rate (∼127%), total GH basal secretion (∼120%), total pulsatile secretion (∼88%), and total GH secretion (∼89%) over the control (i.e., no exercise) condition. GH secretions for the resistance exercise conditions were not different from control. CONCLUSIONS: The fact that the 2-h aerobic exercise condition resulted in higher energy expenditure than the other exercise conditions could offer a partial explanation for the greater GH amplification because of the metabolic effects that GH exerts in stimulating postexercise lipolysis. We conclude that extending the duration of aerobic exercise, but not resistance exercise, from 1- to 2-h significantly amplifies GH secretion during a 20-h period.

Discovery of microRNAs of the stable fly (Diptera: Muscidae) by High-throughput sequencing.

The stable fly, Stomoxys calcitrans (L.), is a serious ectoparasite affecting animal production and health of both animals and humans. Stable fly control relies largely on chemical insecticides; however, the development of insecticide resistance as well as environmental considerations requires continued discovery research to develop novel control technologies. MicroRNAs (miRNAs) are a class of short noncoding RNAs that have been shown to be important regulators of gene expression across a wide variety of organisms, and may provide an innovative approach with regard to development of safer more targeted control technologies. The current study reports discovery ad initial comparative analysis of 88 presumptive miRNA sequences from the stable fly, obtained using high-throughput sequencing of small RNAs. The majority of stable fly miRNAs were 22-23 nt in length. Many miRNAs were arthropod specific, and several mature miRNA sequences showed greater sequence identity to miRNAs from other blood-feeding dipterans such as mosquitoes rather than to Drosophilids. This initial step in characterizing the stable fly microRNAome provides a basis for further analyses of life stage-specific and tissue-specific expression to elucidate their functional roles in stable fly biology.

Identification of ubiquitin-modified lysine residues and novel phosphorylation sites on eukaryotic initiation factor 2B epsilon.

Eukaryotic initiation factor 2Bε (eIF2Bε) plays a critical role in the initiation of mRNA translation and its expression and guanine nucleotide exchange activity are major determinants of the rate of protein synthesis. In this work we provide evidence that the catalytic epsilon subunit of eIF2B is subject to ubiquitination and proteasome-mediated degradation. Lysates of C2C12 myoblasts treated with proteasome inhibitor were subjected to sequential immunoprecipitations for eIF2Bε followed by ubiquitin. Tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated proteins resulted in the identification of five peptides containing ubiquitin (diglycine) modifications on eIF2Bε. The specific lysine residues containing the ubiquitin modifications were localized as Lys-56, Lys-98, Lys-136, Lys-212 and Lys-500 (corresponding to the rat protein sequence). In addition three novel phosphorylation sites were identified including Ser-22, Ser-125, and Thr-317. Moreover, peptides corresponding to the amino acid sequence of the E3 ligase NEDD4 were also detected in the LC-MS/MS analysis, and an interaction between endogenous eIF2Bε with NEDD4 was confirmed by co-immunoprecipitation.

Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): cDNA sequence, baculovirus expression, and biochemical properties.

BACKGROUND: Millions of people and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus Leishmania that are transmitted by several sand fly species. Insecticides are widely used for sand fly population control to try to reduce or interrupt Leishmania transmission. Zoonotic cutaneous leishmaniasis caused by L. major is vectored mainly by Phlebotomus papatasi (Scopoli) in Asia and Africa. Organophosphates comprise a class of insecticides used for sand fly control, which act through the inhibition of acetylcholinesterase (AChE) in the central nervous system. Point mutations producing an altered, insensitive AChE are a major mechanism of organophosphate resistance in insects and preliminary evidence for organophosphate-insensitive AChE has been reported in sand flies. This report describes the identification of complementary DNA for an AChE in P. papatasi and the biochemical characterization of recombinant P. papatasi AChE. METHODS: A P. papatasi Israeli strain laboratory colony was utilized to prepare total RNA utilized as template for RT-PCR amplification and sequencing of cDNA encoding acetylcholinesterase 1 using gene specific primers and 3'-5'-RACE. The cDNA was cloned into pBlueBac4.5/V5-His TOPO, and expressed by baculovirus in Sf21 insect cells in serum-free medium. Recombinant P. papatasi acetylcholinesterase was biochemically characterized using a modified Ellman's assay in microplates. RESULTS: A 2309 nucleotide sequence of PpAChE1 cDNA [GenBank: JQ922267] of P. papatasi from a laboratory colony susceptible to insecticides is reported with 73-83% nucleotide identity to acetylcholinesterase mRNA sequences of Culex tritaeniorhynchus and Lutzomyia longipalpis, respectively. The P. papatasi cDNA ORF encoded a 710-amino acid protein [GenBank: AFP20868] exhibiting 85% amino acid identity with acetylcholinesterases of Cx. pipiens, Aedes aegypti, and 92% amino acid identity for L. longipalpis. Recombinant P. papatasi AChE1 was expressed in the baculovirus system and characterized as an insect acetylcholinesterase with substrate preference for acetylthiocholine and inhibition at high substrate concentration. Enzyme activity was strongly inhibited by eserine, BW284c51, malaoxon, and paraoxon, and was insensitive to the butyrylcholinesterase inhibitors ethopropazine and iso-OMPA. CONCLUSIONS: Results presented here enable the screening and identification of PpAChE mutations resulting in the genotype for insensitive PpAChE. Use of the recombinant P. papatasi AChE1 will facilitate rapid in vitro screening to identify novel PpAChE inhibitors, and comparative studies on biochemical kinetics of inhibition.

Acetylcholinesterases of blood-feeding flies and ticks.

Acetylcholinesterase (AChE) is the biochemical target of organophosphate (OP) and carbamate pesticides for invertebrates, vertebrate nerve agents, and AChE inhibitors used to reduce effects of Alzheimer's disease. Organophosphate pesticides (OPs) are widely used to control blood-feeding arthropods, including biting flies and ticks. However, resistance to OPs in pests affecting animal and human health has compromised control efficacy. OP resistance often results from mutations producing an OP-insensitive AChE. Our studies have demonstrated production of OP-insensitive AChEs in biting flies and ticks. Complementary DNA (cDNA) sequences encoding AChEs were obtained for the horn fly, stable fly, sand fly, and the southern cattle tick. The availability of cDNA sequences enables the identification of mutations, expression and characterization of recombinant proteins, gene silencing for functional studies, as well as in vitro screening of novel inhibitors. The southern cattle tick expresses at least three different genes encoding AChE in their synganglion, i.e. brain. Gene amplification for each of the three known cattle tick AChE genes and expression of multiple alleles for each gene may reduce fitness cost associated with OP-resistance. AChE hydrolyzes the neurotransmitter, acetylcholine, but may have additional roles in physiology and development. The three cattle tick AChEs possess significantly different biochemical properties, and are expressed in neural and non-neural tissues, which suggest separation of structure and function. The remarkable complexity of AChEs in ticks suggested by combining genomic data from Ixodes scapularis with our genetic and biochemical data from Rhipicephalus microplus is suggestive of previously unknown gene duplication and diversification. Comparative studies between invertebrate and vertebrate AChEs could enhance our understanding of structure-activity relationships. Research with ticks as a model system offers the opportunity to elucidate structure-activity relationships for AChE that are important for advances in targeted pest control, as well as potential applications for medicine and biosecurity.

Exercise-induced insulin-like growth factor I system concentrations after training in women.

INTRODUCTION: This study examined the effects of short-term physical training on the acute hormonal response (i.e., growth hormone, total and free insulin-like growth factor I [IGF-I], and IGF binding proteins [IGFBP]-1, IGFBP-2, and IGFBP-3) to resistance exercise (RE) in women. METHODS: Forty-six women (20.3 ± 0.3 yr, mass = 64.1 ± 7.3 kg, height = 165.7 ± 1.0 cm) were randomly assigned to an endurance training (E), resistance training (R), combined training (R + E), or control (C) group for 8wk. Subjects completed a standardized bout of RE (six sets of back squats at 10 repetition maximum) before and after training. Blood samples were obtained at rest (PRE), after the third set, immediately postexercise (POST), and at 15 min and 30 min after exercise. RESULTS: Acute RE significantly increased (P < 0.05) serum growth hormone (mean ± SD; change from PRE to POST = +10.9 ± 7.5 µg·L-1), total IGF-I (+66.1 ± 25.4 µg·L-1), IGFBP-1 (+2.5 ± 3.1 µg·L-1), IGFBP-2 (+86.0 ± 86.8 µg·L-1), and IGFBP-3 (+0.69 ± 0.25 mg·L-1) concentrations and decreased free IGF-I concentrations (-0.14 ± 0.21 µg·L-1). After 8 wk of training, total IGF-I concentrations were significantly increased (change in POST concentrations from week 0 to week 8 = +82.5 ± 120.8 µg·L-1), and IGFBP-1 concentrations were significantly decreased (-6.7 ± 13.6 µg·L-1) during exercise in groups that participated in resistance training (R and R + E); no significant changes were seen after E or C. CONCLUSIONS: Participation in resistance training increased total IGF-I and reduced IGFBP-1 concentrations during acute RE, indicating exercise mode-specific adaptations in the circulating IGF-I system.

Differential regulation of protein synthesis and mTOR signaling in skeletal muscle and visceral tissues of neonatal pigs after a meal.

Protein synthesis (PS) increases after a meal in neonates, but the time course of the changes in PS in different tissues after a meal is unknown. We aimed to evaluate the changes in tissue PS, mammalian target of rapamycin complex 1 (mTORC1) activation, and proportion of ribosomal protein (rp) mRNAs in polysomes over 4 h after a bolus meal in neonatal pigs (n = 6/group; 5- to 7-d-old). The results show a more sustained increase in PS in glycolytic compared with mixed fiber type muscles and no changes in oxidative muscles. PS increased in liver, jejunum, and pancreas but not in kidney and heart. Feeding did not affect AMP-activated protein kinase or RAS-related GTP binding B activation. Phosphorylation of tuberous sclerosis complex 2, proline-rich Akt substrate of 40 kD, mTOR, eukaryotic initiation factor 4E binding protein, and rp S6 kinase 1 increased in all tissues after feeding. The proportion of mRNAs encoding rp S4 and S8 in liver polysomes increased within 30 min postfeeding. These results suggest that feeding stimulates mTORC1 signaling in muscle and viscera, but mTORC1 activation alone is not sufficient to stimulate PS in all tissues.

Simvastatin represses protein synthesis in the muscle-derived C2C12 cell line with a concomitant reduction in eukaryotic initiation factor 2B expression.

Statins are a widely prescribed class of cholesterol lowering drugs whose use is frequently associated with muscle-related ailments. A number of mechanisms have been implicated in statin-induced myotoxicity including alterations in both protein synthesis and protein degradation. The objective of the present study was to explore the mechanism(s) contributing to the statin-induced reduction in protein synthesis in the muscle-derived C2C12 cell line. Cells were treated with 10 µM simvastatin or vehicle alone for 24 h in 1% serum. Cells exposed to simvastatin exhibited reduced rates of protein synthesis, as evidenced by [(35)S]methionine and [(35)S]cysteine incorporation into protein. The reduction in protein synthesis occurred with a concomitant decrease in expression and activity of eukaryotic initiation factor 2B (eIF2B), a regulated and rate-controlling guanine nucleotide exchange factor known to affect global rates of protein synthesis. The reductions in protein synthesis and eIF2B expression were prevented by coincubation with mevalonate. Simvastatin treatment also resulted in a proteasome-sensitive reduction in the protein expression of all the subunits of the eIF2B heteropentameric complex. Finally, increased phosphorylation of the catalytic ε-subunit at Ser(535) was observed, an event consistent with an observed reduction in eIF2B activity. These results suggest that repression of eIF2B expression and activity may contribute, at least in part, to the statin-induced reduction in protein synthesis.

Ectopic expression of eIF2Bepsilon in rat skeletal muscle rescues the sepsis-induced reduction in guanine nucleotide exchange activity and protein synthesis.

Eukaryotic initiation factor 2B (eIF2B) is a guanine nucleotide exchange factor (GEF) whose activity is both tightly regulated and rate-controlling with regard to global rates of protein synthesis. Skeletal muscle eIF2B activity and expression of its catalytic epsilon-subunit (eIF2Bepsilon) have been implicated as potential contributors to the altered rates of protein synthesis in a number of physiological conditions and experimental models. The objective of this study was to directly examine the effects of exogenously expressed eIF2Bepsilon in vivo on GEF activity and protein synthetic rates in rat skeletal muscle. A plasmid encoding FLAG-eIF2Bepsilon was transfected into the tibialis anterior (TA) of one leg, while the contralateral TA received a control plasmid. Ectopic expression of eIF2Bepsilon resulted in increased GEF activity in TA homogenates of healthy rats, demonstrating that the expressed protein was catalytically active. In an effort to restore a deficit in eIF2B activity, we utilized an established model of chronic sepsis in which skeletal muscle eIF2B activity is known to be impaired. Ectopic expression of eIF2Bepsilon in the TA rescued the sepsis-induced deficit in GEF activity and muscle protein synthesis. The results demonstrate that modulation of eIF2Bepsilon expression may be sufficient to correct deficits in skeletal muscle protein synthesis associated with sepsis and other muscle-wasting conditions.

Circulating bioactive and immunoreactive IGF-I remain stable in women, despite physical fitness improvements after 8 weeks of resistance, aerobic, and combined exercise training.

Insulin-like growth factor-I (IGF-I) is regulated by a number of IGF-binding proteins (IGFBPs) and proteases that influence IGF-I bioactivity. A specific IGF-I kinase receptor activation assay (KIRA) has been developed that determines the ability of IGF-I to activate the IGF-I receptor by quantification of intracellular receptor autophosphorylation on IGF-I binding. KIRA-assessed IGF-I bioactivity has not been utilized within the context of chronic exercise training paradigms. This study measured total and free immunoreactive IGF-I, bioactive IGF-I, and IGFBP-1, -2, and -3 before (Pre), during (Mid), and after (Post) 8 wk of exercise training in young, healthy women, who were randomized into one of four groups: control (n = 10), resistance (n = 18), aerobic (n = 13), and combined (n = 15) exercise training. The training programs were effective in improving physical fitness specific to the exercise mode engaged in: increases were observed for lean mass ( approximately 2%), aerobic fitness (6-7%), and upper (20-24%) and lower (15-48%) body strength (all P values < 0.05). By contrast, no time, group, or interaction effects were observed for the circulating IGF-I system, as immunoreactive total (Pre = 264 +/- 16 microg/l; Mid = 268 +/- 17 microg/l; Post = 271 +/- 17 microg/l), free (Pre = 0.70 +/- 0.1 microg/l; Mid = 0.63 +/- 0.1 microg/l; Post = 0.63 +/- 0.2 microg/l) and bioactive (Pre = 2.35 +/- 0.3 microg/l; Mid = 2.25 +/- 0.3 microg/l; Post = 2.33 +/- 0.3 microg/l) IGF-I were unchanged throughout the study. All IGFBP measures were also unchanged. We conclude that increased lean mass, aerobic fitness, and upper and lower body strength resulting from an 8-wk exercise training programs can occur without concomitant increases in either circulating bioactive or immunoreactive IGF-I, as well as associated IGFBPs. In terms of reflecting positive anabolic neuromuscular outcomes, these data do not support a role for endocrine-derived IGF-I.

Effects of exercise mode and duration on 24-h IGF-I system recovery responses.

INTRODUCTION: This study hypothesized that insulin-like growth factor-binding protein (IGFBP), rather than insulin-like growth factor I (IGF-I) itself, would be more responsive to acute exercise stress in a dose-dependent fashion. METHODS: Eight men (24 +/- 5 yr, 87 +/- 9 kg, 182 +/- 6 cm, 21 +/- 5% body fat) had blood drawn every 4 h after exercise for 24 h and assayed for IGF-I, IGFBP-1, -3, -6, the acid labile subunit (ALS), insulin, glucose, and nonesterified free fatty acids on five occasions: no exercise (control, C), moderate-duration resistance exercise (MDRE; 25, 5-10 repetition maximum (RM) sets), long-duration resistance exercise (LDRE; 50, 5-10 RM sets), moderate-duration aerobic exercise (MDAE; three 15-min cycling bouts at approximately 70% (.)VO2peak), and long-duration aerobic exercise (LDAE; six 15-min cycling bouts at approximately 70% (.)VO2peak). Energy requirements were determined from resting metabolic rate, age, and a physical activity factor. Dietary control was implemented by providing all meals during the experimental trials. A two-way ANOVA with repeated measures (P < 0.05) was used for statistical analysis. RESULTS: Significant exercise effects were observed for IGFBP-1 (C: 14.0 +/- 2.7 < MDRE: 35.9 +/- 8.6 = LDRE: 45.2 +/- 10.6 = MDAE: 34.2 +/- 7.4 = LDAE: 47.0 +/- 11.8 ng x mL(-1) and insulin (C: 26.0 +/- 9 < LDRE: 13.2 +/- 6 ng x mL). In addition, a dose-response relationship was observed for the IGFBP-1 response (long-duration exercise (46 +/- 10 ng x mL(-1)) > moderate-duration exercise (35 +/- 7 ng x mL(-1)). There were no exercise effects for total IGF-I, IGFBP-3, and ALS. Effects of time of day were observed for all variables except ALS. CONCLUSIONS: For the circulating IGF-I system components measured, only IGFBP-1 seems to be a sensitive biomarker capable of assessing the physiological strain of acute physical exercise.

Effects of acute and chronic exercise on disulfide-linked growth hormone variants.

PURPOSE: To test the hypothesis that the appearance of disulfide-linked growth hormone (GH) aggregates during and after an acute resistance exercise test (ARET) in men could be influenced by chronic physical training. METHODS: Fourteen men (28 +/- 1 yr) underwent two different 8-wk physical training programs designed to improve military performance. Before and after chronic training, subjects performed an ARET (six sets of 10 repetition-maximum squat) and had venous blood drawn pre-, mid-, and post-ARET (0, 15, and 30 min postexercise). To determine whether GH molecules were disulfide-linked, serum samples were chemically reduced via glutathione (GSH). Serum immunoreactive GH (IRGH) and immunofunctional GH (IFGH) concentrations were determined using two specific immunoassays, in nonreduced (-GSH) and reduced (+GSH) states. Data were analyzed using repeated-measures ANOVA. RESULTS: No differences were observed in the GH responses of the two training programs; therefore, training group data were combined for analysis. GSH reduction increased the mean GH signal (-GSH: 1.4 +/- 0.3 microg x L(-1) vs +GSH: 1.7 +/- 0.3 microg x L(-1); P < 0.01) only when quantifying IRGH. Post hoc testing indicated that serum contained IRGH disulfide-linked GH aggregates at the mid, 0-, 15-, and 30-min posttime points of the ARET (P < 0.01), whereas GSH reduction did not affect IFGH concentrations. Chronic physical training had no effect on the ARET-induced GH response. CONCLUSION: Acute resistance exercise leads to the appearance of disulfide-linked IRGH aggregates, and this response does not appear to be affected by 8 wk of chronic physical training. The physiological significance of increased proportions of disulfide-linked GH aggregates postexercise remains uncertain; however, structural alterations in GH moieties after acute exercise may represent important regulatory steps in mediating GH biological activity at selected target tissues.

Nocturnal growth hormone secretory dynamics are altered after resistance exercise: deconvolution analysis of 12-hour immunofunctional and immunoreactive isoforms.

To characterize the effects of daytime exercise on subsequent overnight growth hormone (GH) secretion and elimination dynamics, serum was sampled, and GH was measured every 10 min for 12 h (1800 to 0600) in a control (CON) condition and after a 50-set resistance exercise protocol (EX) from 1500 to 1700. GH was measured with a conventional immunoreactive (IR) and an immunofunctional (IF) assay, and values were analyzed via a multi-parameter deconvolution analysis. EX resulted in a higher overnight secretory burst frequency [CON: 7.6 (SD 2.4) < EX: 9.4 (2.2) bursts per 12 h, P = 0.005] but lower mean burst mass [CON: 9.2 (4.7) > EX: 6.0 (2.9) microg/l, P = 0.019] and secretory rate [CON: 0.68 (0.29) > EX: 0.48 (0.23) microg/l/min; P = 0.015; ANOVA main effect means presented]. Approximate entropy (ApEn) was greater after EX, indicating a less orderly GH release process than CON. The estimated half-life of IF GH was significantly lower than IR GH [IF: 15.3 (1.1) < IR 19.8 (1.6) min, P < 0.001] but similar between the CON and EX conditions (approximately 17 min). Despite the changes in secretory dynamics, 12-h mean and integrated GH concentrations were similar between conditions. The results suggest that although quantitatively similar total amounts of GH are secreted overnight in CON and EX conditions, resistance exercise alters the dynamics of secretion by attenuating burst mass and amplitude yet increasing burst frequency.

Minimally invasive sampling of transdermal body fluid for the purpose of measuring insulin-like growth factor-I during exercise training.

Insulin-like growth factor-I (IGF-I) is a ubiquitous hormone that is secreted in both an endocrine and an autocrine/paracrine manner. IGF-I has conventionally been measured in serum; however, transdermal body fluid (TDF) remains as an unexplored biocompartment in which IGF-I also resides and may be more biologically relevant because of its proximity to tissues and cells. The purpose of this study was to compare IGF-I in serum versus IGF-I in TDF before and after 8 weeks of physical training. Twenty-eight healthy men (28 +/- 5 years old, 176 +/- 8 cm tall, weighing 83 +/- 11 kg) had TDF obtained by a novel, minimally invasive method that included the application of continuous vacuum pressure on forearm skin perforated with tiny micropores created by a focused beam from a laser system and also had blood obtained by venipuncture. An enzyme-linked immunosorbent assay measured total IGF-I concentrations. A repeated-measures analysis of variance (biocompartment x time) and Pearson Product Moment Correlation coefficients (P < or = 0.05) were used for statistical analyses. Data are presented as mean +/- SE. Total TDF IGF-I was significantly lower than serum IGF-I both before (TDF, 91 +/- 6 ng/mL; serum, 375 +/- 17 ng/mL) and after (TDF, 83 +/- 5 ng/mL; serum, 363 +/- 19 ng/mL) the exercise training. Serum and TDF IGF-I values were not significantly different pre- to post-training. Serum and TDF IGF-I levels were significantly correlated pre-training (r = 0.41), but not post-training (r = 0.34). The percent change between serum and TDF was not correlated (r = 0.09). This study has demonstrated that total IGF-I can be sampled and measured in TDF via a minimally invasive manner and is appreciably (approximately 76%) less than total IGF-I measured in serum. Additionally, the IGF-I measurements in these two biocompartments were not closely associated, possibly indicating an uncoupled, rather than a linked, regulation of IGF-I among the body's biocompartments.

Altered secretion of growth hormone and luteinizing hormone after 84 h of sustained physical exertion superimposed on caloric and sleep restriction.

The pulsatile release of growth hormone (GH) and luteinizing hormone (LH) from the anterior pituitary gland is integral for signaling secretion of insulin-like growth factor (IGF)-I and testosterone, respectively. This study examined the hypothesis that 84 h of sustained physical exertion with caloric and sleep restriction alters the secretion of GH and LH. Ten male soldiers [22 yr (SD 3), 183 cm (SD 7), 87 kg (SD 8)] had blood drawn overnight from 1800 to 0600 every 20 min for GH, LH, and leptin and every 2 h for IGF-I (total and free), IGF binding proteins-1 and -3, testosterone (total and free), glucose, and free fatty acids during a control week and after 84 h of military operational stress. Time-series cluster and deconvolution analyses assessed the secretion parameters of GH and LH. Significant results (P < or = 0.05) were as follows: body mass (-3%), fat-free mass (-2.3%), and fat mass (-7.3%) declined after military operational stress. GH and LH secretion burst amplitude (approximately 50%) and overnight pulsatile secretion (approximately 50%), IGF binding protein-1 (+67%), and free fatty acids (+33%) increased, whereas leptin (-47%), total (-27%) and free IGF-I (-32%), total (-24%) and free testosterone (-30%), and IGF binding protein-3 (-6%) decreased. GH and LH pulse number were unaffected. Because GH and LH positively regulate IGF-I and testosterone, these data imply that the physiological strain induced a certain degree of peripheral resistance. During periods of energy deficiency, amplitude modulation of GH and LH pulses may precede alterations in pulse numbers.

Reliability assessment of ballistic jump squats and bench throws.

The purpose of this investigation was to determine the test-retest reliability and coefficient of variation of 2 novel physical performance tests. Ten healthy men (22.0 +/- 3.0 years, 87.0 +/- 8.0 kg, 20.0 +/- 5.0% body fat) performed 30 continuous and dynamic jump squats (JS) and bench throws (BT) on 4 separate occasions. The movements were performed under loaded conditions utilizing 30% of subject's predetermined 1 repetition maximum in the back squat and bench press. Mean power (MP; W), peak power (PP; W), mean velocity (MV; m.s(-1)), peak velocity (PV; m.s(-1)), and total work (TW; J) were assessed using a ballistic measurement system (Innervations Inc., Muncie, IN). Data were analyzed using repeated measures analysis of variance with Duncan's post hoc test when mean differences were p < or = 0.05. Intraclass correlation coefficient (ICC) and within-subject coefficient of variation (CV%) were also calculated. All values are presented as mean +/- SE. BT variables were statistically similar across the 4 sessions: MP (350.0 +/- 13.9 W), PP (431.4 +/- 18.5 W) MV (1.6 +/- 0.03 m.s(-1)), PV (2.0 +/- 0.03 m.s(-1)), and TW (199.1 +/- 7.2 J). For JS, session 3 PP (1,669.8 +/- 111.2 W) was significantly greater vs. sessions 1, 2, and 4 (1,601.2 +/- 58.4 W). Session 4 MP (1,403.2 +/- 88.6 W) and MV (1.9 +/- 0.1 m.s(-1)) for JS were significantly lower during sessions 1, 2, and 3 (MP: 1,479.4.5 +/- 44.8 W, MV: 2.0 +/- 0.05 m.s(-1)). TW (834.7 +/- 24.3 J) and PV (2.2 +/- 0.04 m.s(-1)) were statistically similar during all sessions for JS. The CVs ranged from 3.0 to 7.6% for the BT and 3.2 to 5.7% for the JS. ICCs for MP, PP, MV, PV, and TW were 0.92, 0.95, 0.94, 0.91, and 0.95, respectively, during BT. ICCs during JS for MP, PP, MV, PV, and TW were 0.96, 0.98, 0.94, 0.94, and 0.89, respectively. The results of the current study support the use of a 30 continuous and dynamic BT protocol as a reliable upper-body physical performance test, which can be administered with minimal practice. Slightly greater variability for JS was observed, although the test had high reliability.

IGF-I system responses during 12 weeks of resistance training in end-stage renal disease patients.

OBJECTIVE: To examine the hypothesis that 12 weeks of resistance training would alter circulating concentrations of IGF-I system components in end-stage renal disease (ESRD) patients. DESIGN: Ten ESRD patients underwent 12 weeks of resistance training after a 6 week control period and had morning fasted blood drawn on four occasions (weeks - 6, 0, 6, 12). Immunoassays were performed for serum total and free IGF-I, IGF binding proteins (IGFBPs) 2 and 3, and the acid labile subunit (ALS). Immunoaffinity depletion of ALS-based complexes allowed measurement of non-ternary (i.e., binary) IGF-I and IGFBP-3. RESULTS: Significant improvements in strength and functional performance were observed. All IGF-I measures were stable during the control period and no changes were observed for the first 6 weeks of resistance training. At week 12, total IGF-I (-15.4+/-28.9%), ternary IGF-I (-16.4+/-36.7%), and the IGF-I/IGFBP-3 ratio had significantly (p < or = 0.05) declined from week 0 values. No changes were observed for free IGF-I, IGFBPs 2 and 3, or the acid labile subunit. The proportion of IGF-I in ternary ( approximately 76.3+/-6.8%), non-ternary ( approximately 22.5+/-6.6%), and free ( approximately 1.2+/-0.5%) forms remained constant throughout the training. CONCLUSIONS: 12 weeks of resistance training in ESRD patients induced a decline in total IGF-I, but did not alter the proportion of IGF-I circulating in free, ternary or non-ternary molecular complexes. The decline in IGF-I occurs in the presence of positive training adaptations on physical performance and we conclude that this response pattern appears to be reflective of favorable neuromuscular anabolic adaptations.

Growth hormone molecular heterogeneity and exercise.

There are more than 100 molecular isoforms of circulating growth hormone (GH), but the traditional measurement approach in the exercise literature has only focused on the main isoform (i.e., 22 kDa). New assay methodologies now can assess various GH isoforms. The current data suggest that exercise results in the preferential release of GH isoforms with extended half-lives, thereby sustaining biological actions.